MiR-646 Suppresses Lung Adenocarcinoma Cell Proliferation,Migration And Invasion By Downregulating EGFR

Background:As one of the most frequently diagnosed cancer and the leading cause of cancer death,lung cancer can be divided into small cell lung cancer?SCLC?and non-small cell lung cancer?NSCLC?.Non-small cell lung cancer?NSCLC?accounts for 85%of all diagnosed lung cancers.NSCLC is currently defined as one of the histological phenotypes including adenocarcinoma,squamous cell carcinoma and large cell carcinoma.The latest data shows that the morbidity of lung adenocarcinoma occupied more than 40%in lung cancer,which requires much more attention.The incidence of lung cancer is extremely hidden and most patients with lung cancer present with metastasis at diagnosis.Although there is a great progress in surgery,radiotherapy and chemotherapy,the 5-year survival rate of lung cancer is less than 16%.Therefore,researchers have intensive interest in molecular studies aiming at early detection and targeted treatment of lung cancer.EGFR as a member of the ErbB receptor tyrosine kinase family plays an essential role in the development and progression of NSCLC.Over-expression,copy number variations or mutations activated of EGFR are correlated with tumor genesis and some important signaling cascades in tumor cells,and are important for prognosis of lung cancer.In view of the important role of EGFR,it has been considered as one of the most promising therapeutic targets in lung cancer.mi RNAs are short,single-stranded,endogenous,non-coding RNA molecules about18-25 nucleotides in length.These small molecules mainly bind imperfectly to the3?untranslated region of target messenger RNAs?mRNAs?.They negatively regulate gene expression post-transcriptionally by inhibiting translation and causing degradation of target mRNA.More than a thousand miRNAs exist in human genome and each one can potentially regulate hundreds of mRNAs.miRNAs play an important part in many cellular processes,such as differentiation,proliferation,apoptosis,and stress response.miRNAs in tumors have a signifcant different expression,which confirms the roles miRNAs played in tumors.miRNAs can trigger many signaling pathways to cause different effects.Based on the signaling pathways,miRNAs can be classifed with two main types:the tumor suppressive miRNAs and oncogenic miRNAs.miRNAs involved in lung cancer have the ability to regulate the tumorigenesis,survival,angiogenesis,migration and invasion of tumor.miR-646,which is known to be mammalian specific,belongs to the miR-15/107 gene group,with the sequence AGCAGC-staring at either the first nucleotide or the second nucleotide from the 5'end of the mature miRNA.miR-646 was first identified in a study of miRNAs expressed in human cerebral cortical gray and white matter.mi R-646 plays important roles in cell division,angiogenesis,and metabolic pathways.The expression of miR-646 is downregulated in many tumors,such as renal cell carcinoma,osteosarcoma and squamous cell carcinoma and miR-646 could be a potential therapeutic target for cancer therapy.However,there was no report about the expression and function of miR-646 in lung adenocarcinoma.Method:Firstly,we detected the expression of miR-646 in lung adenocarcinoma cancer tissues and adjacent tissues by RT-PCR,and statistical analyses of the correlation between miR-646 and clinical pathological characteristics were carried out.After these we detected the content of EGFR in tissues by RT-PCR and western blot,then analyzed the correlation between them by statistical means.Last,we used statistical methods to analyze the correlation between miR-646 and EGFR.Secondly,TargetScan and miRDB software were used to predict target protein of miR-646.Subsequently,we examined the targeting effect of miR-646 on EGFR by luciferase reporter gene assay.Furthermore,we detected the levels of EGFR protein and mRNA in the cells of lung adenocarcinoma cells by using miR-646 mimics or inhibitors to overexpression or repressed expression miR-646.Finally,we used miR-646 mimic or inhibitor to detect the proliferation,invasion and migration of lung adenocarcinoma cells in the presence of high or low expression of miR-646.At the same time,we analyzed how miR-646 regulates the proliferation,invasion and migration of lung cancer cells.Results:Part ?:The expression of miR-646 and EGFR in lung adenocarcinoma tissue and the correlation between themThe expression of miR-646 in lung adenocarcinoma tissue was lower than that in the adjacent tissues.Clinicopathological analysis showed that there were no significant relationship of miR-646 expression in 80 cases of pulmonary adenocarcinoma tissues with age,gender,family history and smoking history,while were negatively correlated with tumor sizes,lymphatic node metastasis and TNM staging of lung cancer.The protein and mRNA expression of EGFR in lung cancer tissue were higher than those in the adjacent tissues.We further found that the expression of miR-646 was negatively correlated with EGFR.Part ?:miR-646 inhibits the expression of EGFR by targeting itTargetScan and miRDB predicted and found that miR-646 can target multiple proteins,and mi R-646 could be combined with 3-UTR of EGFR.The luciferase reporter assay was taken to determine whether EGFR as a potential target gene of miR-646.Results showed that co-transfection of EGFR-WT with mi R-646 got a lower luciferase activity in A549cells,while the cells co-transfected with EGFR-mutate or mi R-646 AS?Antisense?did not observe significant variations.To further validate the miRNA-target interaction,western blot and RT-PCR were taken.Results showed there was a remarkably down regulated of EGFR when cells were transfected with mi R-646 mimic.By contrast,when the cells were transfected with miR-646 inhibitor,the expression of EGFR was upregulated.Part ?:The effect of miR-646 on the lung adenocarcinoma cells by inhibiting the EGFR/AKT signaling pathwayIt was well known that EGFR/Akt pathway was associated with cancer cells proliferation and metastasis.To determine the function of miR-646 to EGFR/Akt pathway in lung adenocarcinoma,we first examined the effect of miR-646 on the cell proliferation by MTT assay.We found the proliferation of A549 was signifcantly blocked by upregulation of the expression of miR-646.On the contrary,when the mi R-646 expression decreased,the cells proliferation had been promoted.Since we knew CDK4 and CDK6were key downstream proteins of EGFR/Akt pathway,we examined the expression of CDK4 and CDK6 by western blot and RT-PCR.Results showed that the expression of EGFR,EGFR^p-Tyr1068,Akt^p-Thr308,CDK4,and CDK6 were negative correlation with the expression of miR-646.All of these were sufficient to explain that miR-646 can negatively regulate EGFR and inhibit A549 cells proliferation by affecting its downstream pathway.To study whether miR-646 was involved in metastasis of tumor cells,transwell assays?with or without matrigel?were performed.Results showed that miR-646 signifcantly depressed the invasion and migration potential of A549.In addition,we found MMP2 was signifcantly downregulated by miR-646 through EGFR/Akt pathway.These results confrmed that miR-646 could restrain the metastasis of lung adenocarcinoma cells by inhibiting EGFR/Akt pathway.Conclusion:1.miR-646 expression was decreased in pulmonary adenocarcinoma tissues and had significant negatively correlation with tumor size,lymphatic node metastasis,TNM staging of lung cancer and with expression of EGFR.2.miR-646 can directly act on EGFR,downregulate the expression of EGFR.3.Overexpression of miR-646 could suppress proliferation,migration and invasion abilities of lung adenocarcinoma cells through EGFR/Akt pathway,which suggests that miR-646 could be a tumor suppressor miRNAs.